Melon line HAR-DV15-4299MO

ABSTRACT

The invention provides seed and plants of melon line HAR-DV15-4299MO. The invention thus relates to the plants, seeds, and tissue cultures of melon line HAR-DV15-4299MO and to methods for producing a melon plant produced by crossing such plants with themselves or with another melon plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to plants, seeds, plant parts, and tissue cultures of melon line HAR-DV15-4299MO comprising introduced beneficial or desirable traits.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of inbred melon line HAR-DV15-4299MO.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety or hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer, including greater yield,resistance to insects and pathogens, tolerance to environmental stress,and nutritional value.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all genetic loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for manygenetic loci. Conversely, a cross of two plants each heterozygous at anumber of loci produces a population of hybrid plants that differgenetically and are not uniform. The resulting non-uniformity makesperformance unpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines and hybrids derivedtherefrom are developed by selfing and selection of desired phenotypes.The new lines and hybrids are evaluated to determine which of those havecommercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a melon plant of the melonline designated HAR-DV15-4299MO. Also provided are melon plants havingall the physiological and morphological characteristics of such a plant.Parts of these melon plants are also provided, for example, includingpollen, an ovule, an embryo, a seed, a scion, a rootstock, a fruit, anda cell of the plant.

In another aspect of the invention, a plant of melon lineHAR-DV15-4299MO comprising an added heritable trait is provided. Theheritable trait may comprise a genetic locus that is, for example, adominant or recessive allele. In one embodiment of the invention, aplant of melon line HAR-DV15-4299MO is defined as comprising a singlelocus conversion. In specific embodiments of the invention, an addedgenetic locus confers one or more traits such as, for example, herbicidetolerance, insect resistance, disease resistance, and modifiedcarbohydrate metabolism. In further embodiments, the trait may beconferred by a naturally occurring gene introduced into the genome of aline by backcrossing, a natural or induced mutation, or a transgeneintroduced through genetic transformation techniques into the plant or aprogenitor of any previous generation thereof. When introduced throughtransformation, a genetic locus may comprise one or more genesintegrated at a single chromosomal location.

In some embodiments, a single locus conversion includes one or moresite-specific changes to the plant genome, such as, without limitation,one or more nucleotide modifications, deletions, or insertions. A singlelocus may comprise one or more genes or nucleotides integrated ormutated at a single chromosomal location. In one embodiment, a singlelocus conversion may be introduced by a genetic engineering technique,methods of which include, for example, genome editing with engineerednucleases (GEEN). Engineered nucleases include, but are not limited to,Cas endonucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); engineered meganucleases,also known as homing endonucleases; and other endonucleases for DNA orRNA-guided genome editing that are well-known to the skilled artisan.

The invention also concerns the seed of melon line HAR-DV15-4299MO. Themelon seed of the invention may be provided as an essentiallyhomogeneous population of melon seed of melon line HAR-DV15-4299MO.Essentially homogeneous populations of seed are generally free fromsubstantial numbers of other seed. Therefore, in some embodiments, seedof melon line HAR-DV15-4299MO may be defined as forming at least about97% of the total seed, including at least about 98%, 99% or more of theseed. The seed population may be separately grown to provide anessentially homogeneous population of melon plants designatedHAR-DV15-4299MO.

In yet another aspect of the invention, a tissue culture of regenerablecells of a melon plant of melon line HAR-DV15-4299MO is provided. Thetissue culture will preferably be capable of regenerating melon plantscapable of expressing all of the physiological and morphologicalcharacteristics of the starting plant, and of regenerating plants havingsubstantially the same genotype as the starting plant. Examples of someof the physiological and morphological characteristics of the melon lineHAR-DV15-4299MO include those traits set forth in the table herein. Theregenerable cells in such tissue cultures may be derived, for example,from embryos, meristems, cotyledons, pollen, leaves, anthers, roots,root tips, pistils, flowers, seed and stalks. Still further, the presentinvention provides melon plants regenerated from a tissue culture of theinvention, the plants having all the physiological and morphologicalcharacteristics of melon line HAR-DV15-4299MO.

In still yet another aspect of the invention, processes are provided forproducing melon seeds, plants and fruit, which processes generallycomprise crossing a first parent melon plant with a second parent melonplant, wherein at least one of the first or second parent melon plantsis a plant of melon line HAR-DV15-4299MO. These processes may be furtherexemplified as processes for preparing hybrid melon seed or plants,wherein a first melon plant is crossed with a second melon plant of adifferent, distinct genotype to provide a hybrid that has, as one of itsparents, a plant of melon line HAR-DV15-4299MO. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent melon plant, oftenin proximity so that pollination will occur for example, mediated byinsect vectors. Alternatively, pollen can be transferred manually. Wherethe plant is self-pollinated, pollination may occur without the need fordirect human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent melon plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the flowers (i.e., killing or removing the pollen).

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent melon plants. Yet another step comprisesharvesting the seeds from at least one of the parent melon plants. Theharvested seed can be grown to produce a melon plant or hybrid melonplant.

The present invention also provides the melon seeds and plants producedby a process that comprises crossing a first parent melon plant with asecond parent melon plant, wherein at least one of the first or secondparent melon plants is a plant of melon line HAR-DV15-4299MO. In oneembodiment of the invention, melon seed and plants produced by theprocess are first generation (F₁) hybrid melon seed and plants producedby crossing a plant in accordance with the invention with another,distinct plant. The present invention further contemplates plant partsof such an F₁ hybrid melon plant, and methods of use thereof. Therefore,certain exemplary embodiments of the invention provide an F₁ hybridmelon plant and seed thereof.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from melon line HAR-DV15-4299MO, the methodcomprising the steps of: (a) preparing a progeny plant derived frommelon line HAR-DV15-4299MO, wherein said preparing comprises crossing aplant of the melon line HAR-DV15-4299MO with a second plant; and (b)crossing the progeny plant with itself or a second plant to produce aseed of a progeny plant of a subsequent generation. In furtherembodiments, the method may additionally comprise: (c) growing a progenyplant of a subsequent generation from said seed of a progeny plant of asubsequent generation and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and repeating the steps for anadditional 3-10 generations to produce a plant derived from melon lineHAR-DV15-4299MO. The plant derived from melon line HAR-DV15-4299MO maybe an inbred line, and the aforementioned repeated crossing steps may bedefined as comprising sufficient inbreeding to produce the inbred line.In the method, it may be desirable to select particular plants resultingfrom step (c) for continued crossing according to steps (b) and (c). Byselecting plants having one or more desirable traits, a plant derivedfrom melon line HAR-DV15-4299MO is obtained which possesses some of thedesirable traits of the line/hybrid as well as potentially otherselected traits.

In certain embodiments, the present invention provides a method ofproducing food or feed comprising: (a) obtaining a plant of melon lineHAR-DV15-4299MO, wherein the plant has been cultivated to maturity, and(b) collecting at least one melon from the plant.

In still yet another aspect of the invention, the genetic complement ofmelon line HAR-DV15-4299MO is provided. The phrase “genetic complement”is used to refer to the aggregate of nucleotide sequences, theexpression of which sequences defines the phenotype of, in the presentcase, a melon plant, or a cell or tissue of that plant. A geneticcomplement thus represents the genetic makeup of a cell, tissue orplant, and a hybrid genetic complement represents the genetic make-up ofa hybrid cell, tissue or plant. The invention thus provides melon plantcells that have a genetic complement in accordance with the melon plantcells disclosed herein, and seeds and plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that melon line HAR-DV15-4299MO could be identified byany of the many well-known techniques such as, for example, SimpleSequence Length Polymorphisms (SSLPs) (Williams et al., Nucleic AcidsRes., 1 8:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs),DNA Amplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by melon plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a melon plant of the invention with a haploid geneticcomplement of a second melon plant, preferably, another, distinct melonplant. In another aspect, the present invention provides a melon plantregenerated from a tissue culture that comprises a hybrid geneticcomplement of this invention.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of the mean for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive. When used in conjunctionwith the word “comprising” or other open language in the claims, thewords “a” and “an” denote “one or more,” unless specifically notedotherwise. The terms “comprise,” “have” and “include” are open-endedlinking verbs. Any forms or tenses of one or more of these verbs, suchas “comprises,” “comprising,” “has,” “having,” “includes” and“including,” are also open-ended. For example, any method that“comprises,” “has” or “includes” one or more steps is not limited topossessing only those one or more steps and also covers other unlistedsteps. Similarly, any plant that “comprises,” “has” or “includes” one ormore traits is not limited to possessing only those one or more traitsand covers other unlisted traits.

Other objects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds, and derivatives of melon line HAR-DV15-4299MO. This line showsuniformity and stability within the limits of environmental influencefor the traits described hereinafter. A small percentage of variants canoccur within commercially acceptable limits for almost anycharacteristic during the course of repeated multiplication. However novariants are expected. Melon line HAR-DV15-4299MO provides sufficientseed yield. By crossing with a distinct second plant, uniform F₁ progenycan be obtained.

Melon line HAR-DV15-4299MO is a monoecious cantaloupe line that producesa fruit having orange flesh, silver skin, and an extended shelf life.

A. Physiological and Morphological Characteristics of Melon LineHAR-DV15-4299MO

In accordance with one aspect of the present invention, there areprovided plants having the physiological and morphologicalcharacteristics of melon line HAR-DV15-4299MO. Descriptions of thephysiological and morphological characteristics of such plants arepresented in the table that follows.

TABLE 1 Physiological and Morphological Characteristics of Melon LineHAR-DV15-4299MO HAR-DV15- CHARACTERISTIC 4299MO Caribbean Gold Typecommon or common or summer summer Seedling length of hypocotyl (justbefore short long development of the first true leaf) size of cotyledonsmall large intensity of green color of medium dark cotyledon Leaf(mature blade of third leaf) shape reniform reniform lobes shallowlylobed shallowly lobed color dark green dark green RHS Color Chart Value147A 147A length (mm) 82.60 131.40 width (mm) 133.60 160.60 surfacepubescent pubescent Leaf Blade (fully developed but not old leaves,between the 5^(th) and 8^(th) node when the plant has at least 11 nodes)size small large intensity of green color dark dark development of lobesmedium weak length of terminal lobe medium short dentation of marginweak weak blistering strong medium Petiole attitude semi-erecthorizontal length medium long Plant sex expression (at full flowering)monoecious monoecious habit vine vine time of male flowering mediummedium time of female flowering medium medium Young Fruit (green, unripefruit before color change) hue of green color of skin green greenintensity of green color of skin medium light density of dots absent orvery absent or very sparse sparse contrast of dot color/ground color n/aweak conspicuousness of groove absent or very absent or very coloringweak weak intensity of groove coloring n/a light length of pedunclemedium medium thickness of peduncle 1 cm from thin thin fruit extensionof darker area around small small peduncle time of ripening late lateFruit change of skin color from young late in fruit late in fruit fruitto maturity development development length medium very long length (atedible maturity) (cm) 14.62 24.97 diameter broad broad diameter (atedible maturity) 14.29 14.31 (cm) ratio length/diameter medium largeweight (at edible maturity) (gm) 1584.53 1604.80 position of maximumdiameter at middle at middle shape oval oval shape in longitudinalsection broad elliptic circular/round surface (at edible maturity)netted netted blossom scar (at edible maturity) conspicuous obscure ribpresence (at edible maturity) absent absent shipping quality (at ediblefair excellent maturity) abscission (at edible maturity) when ripe whenoverripe maturity (number of days from 90 90 seeding to harvest) groundcolor of skin green green intensity of ground color of skin light lighthue of ground color of skin greenish greenish density of dots absent orvery absent or very sparse sparse density of patches absent or veryabsent or very sparse sparse warts absent absent strength of attachmentof medium medium peduncle at maturity shape of base truncate roundedshape of apex truncate rounded size of pistil scar small medium groovesabsent or very absent or very weakly weakly expressed expressed creasingof surface absent or very absent or very weak weak cork formationpresent present thickness of cork layer very thin very thick pattern ofcork formation netted only netted only density of pattern of cork densevery dense formation rate of change of skin color from absent or veryabsent or very maturity to over maturity slow slow width of flesh inlongitudinal medium thick section (at position of maximum fruitdiameter) main color of flesh orange orange intensity of orange color offlesh light light (only varieties with main color of flesh orange)firmness of flesh firm medium Flesh color near cavity VISUAL (at 07 07edible maturity) RHS Color Chart Value 24C 24C color in center VISUAL(at 07 07 edible maturity) RHS Color Chart Value 24D 24D color near rindVISUAL (at 07 07 edible maturity) RHS Color Chart Value 24D 24Drefractometer % soluble solids 21.97% 19.14% (center of flesh) aroma (atedible maturity) absent absent flavor (at edible maturity) mild somewhatspicy Seed Cavity shape in cross section triangular circular Seed (fullydeveloped and dry seeds, after washing and drying in the shade) lengthshort short width narrow narrow shape pine-nut shape not pine-nut shapecolor cream yellow whitish intensity of color medium n/a number of seedsper fruit 440.00 550.20 grams per 1,000 seeds (gm) 20.25 25.90 Rind netabundant abundant texture firm hard distribution covers entire coversentire fruit fruit coarseness medium coarse very coarse interlacingcomplete complete interstices shallow deep thickness at medial (mm) 1.311.62 primary color VISUAL (at edible 10 06 04 06 maturity) RHS ColorChart Value 194 A 144A net color VISUAL (at edible 10 04 10 01 maturity)RHS Color Chart Value 160D 156B primary color VISUAL (at full 10 06 0406 maturity) RHS Color Chart Value 194 A 144A net color VISUAL (at full10 04 10 01 maturity) RHS Color Chart Value 160D 156B These are typicalvalues. Values may vary due to environment. Values that aresubstantially equivalent are within the scope of the invention.

These are typical values. Values may vary due to environment. Valuesthat are substantially equivalent are within the scope of the invention.

B. Breeding Melon Plants

One aspect of the current invention concerns methods for producing seedof uniform F₁ progeny involving crossing melon line HAR-DV15-4299MO witha second distinct plant. Alternatively, in other embodiments of theinvention, HAR-DV15-4299MO may be crossed with itself or with any secondplant. Such methods can be used for propagation of the melon lineHAR-DV15-4299MO or can be used to produce plants that are derived fromthe melon line HAR-DV15-4299MO. Plants derived from the melon lineHAR-DV15-4299MO may be used, in certain embodiments, for the developmentof new melon varieties.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing melon line HAR-DV15-4299MO with a secondplant followed by multiple generations of breeding according to suchwell-known methods. New varieties may be created by crossing with anysecond plant. In selecting such a second plant to cross for the purposeof developing novel lines, it may be desired to choose those plantswhich either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Once initial crosses have been made, inbreeding andselection take place to produce new varieties. For development of auniform line, often five or more generations of selfing and selectionare involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the invention and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny have the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The plants of the present invention are particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the plants. In selecting a second plant to cross withmelon line HAR-DV15-4299MO for the purpose of developing novel melonlines, it will typically be preferred to choose those plants whicheither themselves exhibit one or more selected desirable characteristicsor which exhibit the desired characteristic(s) when in hybridcombination. Examples of desirable traits may include, in specificembodiments, high seed yield, high seed germination, seedling vigor,high fruit yield, disease tolerance or resistance, adaptability for soiland climate conditions, and delayed fruit ripening. Consumer-driventraits, such as a fruit shape, color, texture, and taste are otherexamples of traits that may be incorporated into new lines of melonplants developed by this invention.

C. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein areprovided modified to include at least a first desired heritable trait.Such plants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of themorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those melon plants which are developed by a plantbreeding technique called backcrossing or by genetic engineering,wherein essentially all of the morphological and physiologicalcharacteristics of a variety are recovered or conserved in addition tothe single locus introduced into the variety via the backcrossing orgenetic engineering technique, respectively. By essentially all of themorphological and physiological characteristics, it is meant that thecharacteristics of a plant are recovered or conserved that are otherwisepresent when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing, introduction of atransgene, or application of a genetic engineering technique.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalmelon plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental melon plant to whichthe locus or loci from the nonrecurrent parent are transferred is knownas the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a melon plant isobtained wherein essentially all of the morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred locus from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny melon plants of a backcross in which a plantdescribed herein is the recurrent parent comprise (i) the desired traitfrom the non-recurrent parent and (ii) all of the physiological andmorphological characteristics of melon the recurrent parent asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

New varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiol., 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,herbicide resistance, resistance to bacterial, fungal, or viral disease,insect resistance, modified fatty acid or carbohydrate metabolism, andaltered nutritional quality. These comprise genes generally inheritedthrough the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. For this selection process, the progeny of the initialcross are assayed for viral resistance and/or the presence of thecorresponding gene prior to the backcrossing. Selection eliminates anyplants that do not have the desired gene and resistance trait, and onlythose plants that have the trait are used in the subsequent backcross.This process is then repeated for all additional backcross generations.

Selection of melon plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection are well known in the art. Suchmethods will be of particular utility in the case of recessive traitsand variable phenotypes, or where conventional assays may be moreexpensive, time consuming or otherwise disadvantageous. In addition,marker assisted selection may be used to identify plants comprisingdesirable genotypes at the seed, seedling, or plant stage, to identifyor assess the purity of a cultivar, to catalog the genetic diversity ofa germplasm collection, and to monitor specific alleles or haplotypeswithin an established cultivar.

Types of genetic markers which could be used in accordance with theinvention include, but are not necessarily limited to, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In particular embodiments of the invention, marker assisted selection isused to increase the efficiency of a backcrossing breeding scheme forproducing a melon line comprising a desired trait. This technique iscommonly referred to as marker assisted backcrossing (MABC). Thistechnique is well-known in the art and may involve, for example, the useof three or more levels of selection, including foreground selection toidentity the presence of a desired locus, which may complement orreplace phenotype screening protocols; recombinant selection to minimizelinkage drag; and background selection to maximize recurrent parentgenome recovery.

D. Plants Derived by Genetic Engineering

Various genetic engineering technologies have been developed and may beused by those of skill in the art to introduce traits in plants. Incertain aspects of the claimed invention, traits are introduced intomelon plants via altering or introducing a single genetic locus ortransgene into the genome of a recited variety or progenitor thereof.Methods of genetic engineering to modify, delete, or insert genes andpolynucleotides into the genomic DNA of plants are well-known in theart.

In specific embodiments of the invention, improved melon lines can becreated through the site-specific modification of a plant genome.Methods of genetic engineering include, for example, utilizingsequence-specific nucleases such as zinc-finger nucleases (see, forexample, U.S. Pat. Appl. Pub. No. 2011-0203012); engineered or nativemeganucleases; TALE-endonucleases (see, for example, U.S. Pat. Nos.8,586,363 and 9,181,535); and RNA-guided endonucleases, such as those ofthe CRISPR/Cas systems (see, for example, U.S. Pat. Nos. 8,697,359 and8,771,945 and U.S. Pat. Appl. Pub. No. 2014-0068797). One embodiment ofthe invention thus relates to utilizing a nuclease or any associatedprotein to carry out genome modification. This nuclease could beprovided heterologously within donor template DNA for templated-genomicediting or in a separate molecule or vector. A recombinant DNA constructmay also comprise a sequence encoding one or more guide RNAs to directthe nuclease to the site within the plant genome to be modified. Furthermethods for altering or introducing a single genetic locus include, forexample, utilizing single-stranded oligonucleotides to introduce basepair modifications in a melon plant genome (see, for example Sauer etal., Plant Physiol, 170(4):1917-1928, 2016).

Methods for site-directed alteration or introduction of a single geneticlocus are well-known in the art and include those that utilizesequence-specific nucleases, such as the aforementioned, or complexes ofproteins and guide-RNA that cut genomic DNA to produce a double-strandbreak (DSB) or nick at a genetic locus. As is well-understood in theart, during the process of repairing the DSB or nick introduced by thenuclease enzyme, a donor template, transgene, or expression cassettepolynucleotide may become integrated into the genome at the site of theDSB or nick. The presence of homology arms in the DNA to be integratedmay promote the adoption and targeting of the insertion sequence intothe plant genome during the repair process through homologousrecombination or non-homologous end joining (NHEJ).

In another embodiment of the invention, genetic transformation may beused to insert a selected transgene into a plant of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plantsthat are well-known to those of skill in the art and applicable to manycrop species include, but are not limited to, electroporation,microprojectile bombardment, Agrobacterium-mediated transformation, anddirect DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

An efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target cells. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates.Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Nat. Biotechnol., 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S.Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, for example,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13:344-348, 1994) and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for plant gene expressioninclude, but are not limited to, the cauliflower mosaic virus (CaMV)P-35S promoter, which confers constitutive, high-level expression inmost plant tissues (see, for example, Odel et al., Nature, 313:810,1985), including in monocots (see, for example, Dekeyser et al., PlantCell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389,1990); a tandemly duplicated version of the CaMV 35S promoter, theenhanced 35S promoter (P-e35S); the nopaline synthase promoter (An etal., Plant Physiol., 88:547, 1988); the octopine synthase promoter(Fromm et al., Plant Cell, 1:977, 1989); and the figwort mosaic virus(P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and an enhancedversion of the FMV promoter (P-eFMV) where the promoter sequence ofP-FMV is duplicated in tandem; the cauliflower mosaic virus 19Spromoter; a sugarcane bacilliform virus promoter; a commelina yellowmottle virus promoter; and other plant DNA virus promoters known toexpress in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can alsobe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., PlantPhysiol., 88:965, 1988), (2) light (for example, pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4)wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to plants of thisinvention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a melon plant according to the invention.Non-limiting examples of particular genes and corresponding phenotypesone may choose to introduce into a melon plant include one or more genesfor insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pesttolerance such as genes for fungal disease control, herbicide tolerancesuch as genes conferring glyphosate tolerance, and genes for qualityimprovements such as yield, nutritional enhancements, environmental orstress tolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s). For example, structuralgenes would include any gene that confers insect tolerance including butnot limited to a Bacillus insect control protein gene as described in WO99/31248, herein incorporated by reference in its entirety, U.S. Pat.No. 5,689,052, herein incorporated by reference in its entirety, U.S.Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference intheir entirety. In another embodiment, the structural gene can confertolerance to the herbicide glyphosate as conferred by genes including,but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPSgene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety, or glyphosate oxidoreductasegene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporatedby reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

E. Definitions

In the description and table herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a genetic locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions or transgenes from one geneticbackground into another.

Crossing: The mating of two parent plants.

Cross-Pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Regeneration: The development of a plant from tissue culture.

Royal Horticultural Society (RHS) Color Chart Value: The RHS Color Chartis a standardized reference which allows accurate identification of anycolor. A color's designation on the chart describes its hue, brightnessand saturation. A color is precisely named by the RHS Color Chart byidentifying the group name, sheet number, and letter, e.g.,Yellow-Orange Group 19A or Red Group 41B.

Self-Pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing or genetic engineering ofa locus, wherein essentially all of the morphological and physiologicalcharacteristics of a melon variety are recovered in addition to thecharacteristics of the single locus.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a melon plant by transformation or sitespecific modification.

F. Deposit Information

A deposit of at least 625 seeds of melon line HAR-DV15-4299MO, disclosedabove and recited in the claims, has been made with theProvasoli-Guillard National Center for Marine Algae and Microbiota(NCMA), 60 Bigelow Drive, East Boothbay, Me., 04544 USA. The date ofdeposit for melon line HAR-DV15-4299MO is Jul. 30, 2021. The accessionnumber for those deposited seeds of melon line HAR-DV15-4299MO is NCMAAccession Number 202107027. Upon issuance of a patent, all restrictionsupon the deposit will be removed, and the deposit is intended to meetall of the requirements of 37 C.F.R. §§ 1.801-1.809. The deposit hasbeen accepted under the Budapest Treaty and will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced if necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed:
 1. A melon plant comprising at least a first set of thechromosomes of melon line HAR-DV15-4299MO, a sample of seed of said linehaving been deposited under NCMA Accession Number
 202107027. 2. A melonseed that produces the plant of claim
 1. 3. The plant of claim 1,wherein the plant is an inbred plant of said melon line HAR-DV15-4299MO.4. The plant of claim 1, wherein the plant is a hybrid melon plant. 5.The seed of claim 2, wherein the seed is an inbred seed of said melonline HAR-DV15-4299MO.
 6. The seed of claim 2, wherein the seed is ahybrid seed.
 7. A plant part of the plant of claim 1, wherein the plantpart comprises a cell of said plant.
 8. A melon plant having all thephysiological and morphological characteristics of the plant of claim 3.9. A tissue culture of regenerable cells of the plant of claim
 1. 10. Amethod of vegetatively propagating the melon plant of claim 1, themethod comprising the steps of: (a) collecting tissue capable of beingpropagated from the plant of claim 1; and (b) propagating a melon plantfrom said tissue.
 11. A method of introducing a trait into a melon line,the method comprising: (a) utilizing as a recurrent parent the plant ofclaim 3 by crossing said plant with a donor melon plant that comprises atrait to produce F₁ progeny; (b) selecting an F₁ progeny that comprisesthe trait; (c) backcrossing the selected F₁ progeny with a plant of thesame melon line used as the recurrent parent in step (a) to producebackcross progeny; (d) selecting a backcross progeny comprising thetrait and the morphological and physiological characteristics of therecurrent parent melon line used in step (a); and (e) repeating steps(c) and (d) three or more times to produce selected fourth or higherbackcross progeny.
 12. A melon plant produced by the method of claim 11,wherein said plant comprises the trait and otherwise comprises all ofthe morphological and physiological characteristics of melon lineHAR-DV15-4299MO.
 13. A method of producing a melon plant comprising anadded trait, the method comprising introducing a transgene conferringthe trait into the plant of claim
 1. 14. A melon plant produced by themethod of claim 13, wherein said plant comprises the trait and otherwisecomprises all of the morphological and physiological characteristics ofmelon line HAR-DV15-4299MO.
 15. A melon plant comprising at least afirst set of the chromosomes of melon line HAR-DV15-4299MO, a sample ofseed of said line having been deposited under NCMA Accession Number202107027, further comprising a transgene.
 16. The plant of claim 15,wherein the transgene confers a trait selected from the group consistingof male sterility, herbicide tolerance, insect resistance, pestresistance, disease resistance, modified fatty acid metabolism,environmental stress tolerance, modified carbohydrate metabolism, andmodified protein metabolism.
 17. A melon plant comprising at least afirst set of the chromosomes of melon line HAR-DV15-4299MO, a sample ofseed of said line having been deposited under NCMA Accession Number202107027, further comprising a single locus conversion.
 18. The plantof claim 17, wherein the single locus conversion confers a traitselected from the group consisting of male sterility, herbicidetolerance, insect resistance, pest resistance, disease resistance,modified fatty acid metabolism, environmental stress tolerance, modifiedcarbohydrate metabolism, and modified protein metabolism.
 19. A methodfor producing a seed of a melon plant derived from melon lineHAR-DV15-4299MO, the method comprising the steps of: (a) crossing themelon plant of claim 1 with itself or a second melon plant; and (b)allowing seed of a line HAR-DV15-4299MO-derived melon plant to form. 20.A method of producing a seed of a line HAR-DV15-4299MO-derived melonplant, the method comprising the steps of: (a) producing a lineHAR-DV15-4299MO-derived melon plant from a seed produced by crossing themelon plant of claim 1 with itself or a second melon plant; and (b)crossing the line HAR-DV15-4299MO-derived melon plant with itself or adifferent melon plant to obtain a seed of a further lineHAR-DV15-4299MO-derived melon plant.
 21. The method of claim 20, themethod further comprising repeating said producing and crossing steps of(a) and (b) using the seed from said step (b) for at least onegeneration to produce a seed of an additional lineHAR-DV15-4299MO-derived melon plant.
 22. A method of producing a melonfruit, the method comprising: (a) obtaining the plant of claim 1,wherein the plant has been cultivated to maturity; and (b) collecting amelon fruit from the plant.